In EP 93912877.3 and U.S. Pat. No. 5,633,284 and their equivalents the applicant disclosed that dermatological or topical compositions comprising the combination of nitrous oxide [N2O] and at least one fatty acid, or lower alkyl ester thereof in a dermatologically acceptable carrier medium are useful in the treatment of a variety of skin, muscle and joint disorders. It also disclosed therein that such combinations may beneficially also include additional active ingredients.
The following active ingredients are specifically mentioned in this regard: coal tar solution, collagen, nicotinamide, nicotinic acid, lanolin, vitamin E, methyl salicylate, arnica and H1-antagonist antihistamines of which only diphenhydramine chloride is specifically mentioned.
Known anti-bacterial, anti-viral or anti-fungal agents were not amongst the active ingredients specifically mentioned in the patents but mention was made therein that coal tar solution (also known as Liquor Picis Carbonis) may be used (as a supplementary active ingredient and that the resultant preparation is suitable for use in the treatment of, inter alia fever blisters, herpes simplex, shingles and chicken pox. While all of these conditions are caused by viral infections, the disclosures in these patents do not refer to that fact.
It is also disclosed in these patents that, in addition to the coal tar solution the composition may also contain an H1-antagonist antihistamine (e.g. diphenhydramine hydrochloride) and may in that form be used in the treatment of atopic and allergic conditions manifesting in skin irritations such as eczema, dermatitis and ringworm. The latter of these conditions is caused by a fungal infection. Again the disclosures in issue do not refer specifically to that fact.
It further disclosed an alternative composition in which the coal tar solution formulation is further provided with collagen and lanolin and this formulation was found to be useful in the treatment of persons suffering from acne vulgaris. Bacteria are involved in the condition but no mention was made of such involvement in those patents.
Since coal tar solution is in itself not known to be an anti-viral, anti-fungal or antibacterial agent, and has merely been mentioned as being weakly antiseptic, the aforementioned disclosures would not have been understood as suggesting that the nitrous oxide and fatty acid combination has any beneficial effect on the anti-viral or anti-fungal or anti-bacterial activity of any recognised anti-viral or anti-fungal or anti-bacterial agent or to have disclosed that such properties are displayed by coal tar solution. As will appear below the enhancement of the anti-bacterial, anti-fungal or anti-viral properties of known agents lie at the very heart of this invention.
Within the context of the disclosure in the abovementioned patent family the notional addressee most likely would, as did the inventor, have understood the role of the coal tar solution to sooth the itching and to assist in the repairing and healing of the skin which was damaged as a result of the infections/conditions in issue.
No agent known for having an effect on the central or peripheral nervous system, was amongst the active ingredients specifically mentioned in these patents. Mention was made therein that coal tar solution (also known as Liquor Picis Carbonis) may be used as a supplementary active ingredient and that the resultant preparation is suitable for use in the treatment, inter alia, of psoriasis, eczemaceous conditions and dermatitis. It is also disclosed in these patents that, in addition to the coal tar solution the composition may also contain an H1-antagonist antihistamine (e.g. diphenhydramine hydrochloride) and that such a preparation may beneficially be used in the treatment of atopic and allergic conditions manifesting in skin irritations such as eczema and dermatitis. The aforementioned conditions have since the date of the above patents come to be regarded by some writers on the topic as manifestations in the skin of underlying disorders which have or include a neurological, or immunological, or some even suggest a neuro-immunological cause. These patents however do not refer to that explanation. It further gives no hint that the effects obtained are the result of any form of systemic operation of any of the components of the formulation. Coal tar liquid is not known to be an agent acting on the central nervous system. Insofar as these patents may be seen to proffer any explanation at all for the results obtained, which is not what the applicant contends for, it is suggested that such explanation would, within the context of the disclosures therein, point to a local effect of soothing irritation and of repairing of skin and associated tissue damage as a result of the conditions mentioned therein. These are psoriasis, shingles, fever blisters, chicken pox, acne, chilblains, eczema, chloasmas, alopecia, dermatitis, ringworm and burn wounds.
The concept of introducing heterologous genetic material into the cells of an organism for the purpose of allowing such genetic material to be incorporated into the cellular DNA of such organism is well known. It is now routinely exercised in vitro and various methods have been developed for the introduction of such heterologous genetic material into the cells of an organism.
The most established of these methods is commonly referred to as the gene gun technique. By this technique genetic material is quite literally shot into the cells of the organism. While some of the cells do not survive the blast, those that do survive are caused to proliferate. Some of these proliferating survivors will have incorporated into its own genome the genetic material shot into it in the form of the DNA. The gene so incorporated may eventually be expressed to yield the product coded for by the DNA so introduced into the organism. It is also known to make use of other forms of vectors by which a gene which is required to be expressed is introduced into an organism. Such vectors include viruses.
While these techniques work adequately in the appropriate in vitro environment on lower organisms such as bacteria, they are not regarded as being generally suitable for implementation in vivo for the introduction of genetic material into a living animal, such as man. The harshness of the gene gun technique by its very nature renders that technique generally unsuitable for in vivo application. It is further generally regarded to be advisable not to expose animals or human beings unnecessarily to viral infections, let alone where such infections are by transgenic viruses, the full genetic nature and potential for mutation of which may be unknown.
There has thus been a long-felt need for an appropriate process by which genetic material may be introduced into selected cells there to express and yield a desired medicinally active substance.
This need is sought to be addressed, inter alia by the disclosure contained in U.S. Pat. No. 6,258,789 (German, et al.) and the patents cited during the examination thereof. German et al discloses a method of delivering a secreted protein into the bloodstream of a mammalian subject by introducing into the gastro-intestinal tract of a mammalian subject, by oral administration, a construct comprising a nucleic acid molecule encoding the desired secreted protein and a promoter sequence operably linked to the nucleic acid molecule, wherein said construct is not packaged in a viral particle, and wherein the method involves introducing the nucleic acid construct into the intestinal epithelial cells of the animal in question so that secretion of the protein takes place at that locus to be available for absorption into the body from the digestive tract.
No agent based on nucleic acid was amongst the active ingredients specifically mentioned in the patents mentioned above.
In WO97/17978 and U.S. Pat. No. 6,221,377 and in corresponding patents and pending patent applications in other jurisdictions the present applicant disclosed that the action of analgesic, anti-inflammatory and anti-pyretic drugs may be enhanced by administering such drugs in conjunction with a medium which comprises nitrous oxide and at least one long chain fatty acid selected from the group consisting of oleic acid, linoleic acid, alpha-linolenic acid, gamma linolenic acid, arachidonic acid, and any of the C1 to C6 alkyl esters of such long chain fatty acids, mixtures of such acids and mixtures of such esters. The medium may comprise the mixture known as Vitamin F Ethyl Ester and may optionally further comprise eicosapentaenoic acid [C20: 5ω3] and decosahexaenoic acid [C22: 6ω3].
While these applications do disclose a systemic effect in that the formulations are, for example, topically applied or orally administered to have an effect, say, in an affected joint or muscle, the specification again fails to disclose the mode or route of transport of the active ingredient from its site of administration to its locus of effect, and speculation in this regard would probably be based on ex post facto hindsight.
It has now surprisingly been found that the aforesaid medium and media related thereto has the ability remarkably to enhance the action of known anti-infective agents. The expression “anti-infective agents” as used herein is intended to have its extended meaning and to include the antimicrobial agents, the anthelmintic agents and the anti-ectoparasitic agents, but to exclude coal tar solution and H1-antagonist antihistamines.
The exclusion of coal tar solution and H1-antagonist antihistamines from the ambit of the present invention is introduced without thereby conceding that the aforementioned patents and applications contain any disclosure of any anti-infective agent properties of such excluded compounds, or that such properties are obvious in the light of the disclosures in such patents or applications. Such inferences are specifically denied. The exclusion is introduced simply to avoid what is anticipated to be a potential obstacle to the grant of a patent in respect of an insignificant part of potential subject matter which part in itself is not considered worth contesting during examination as it might unduly delay the implementation in practice of the significant features of the present invention. It is expected that the remaining bulk of the subject matter of the present invention will greatly contribute to the accessibility of medicines for the treatment of a large range of infections, including secondary infections in HIV-compromised patients, at significantly reduced costs.
The expressions “anthelmintic agents” and “anti-ectoparasitic” agents are further intended to cover both agents which serve to destroy and those which serve to inhibit the proliferation of helminths or ecto-parasites. Those expressions are hence also intended to be understood in the wider sense of these terms. The expression “antimicrobial agents” is similarly intended to be understood in the wider sense of that word and hence to have the meaning ascribed thereto in The McGraw-Hill Dictionary of Scientific and Technical Terms 2nd Ed 1978, namely all chemical compounds that either destroy or inhibit the growth of microscopic and sub microscopic organisms. This term is further specifically intended to include all the compounds falling within the Pharmacological Classification 20 set out as part of Regulation 5(1) of the General Regulations made in terms of the South African Medicines and Related Substances Control Act, Act 101 of 1965, as well as the active ingredients of all products falling within class 18 of the pharmacological classification employed in the Monthly Index of Medical Specialities (“MIMS”) published by Times Media in South Africa. It is thus intended to include:
the anti-bacterial agents (including both antibiotics and substances other than antibiotics such as the sulfonamides, the erythromycins and other macrolides, the aminoglycocides, the tetracyclines, the chloramphenicols and the quinolones);
the anti-fungal agents;
the anti-viral agents (including anti-retroviral agents);
the anti-protozoal agents;
the tuberculostatics;
the anti-leprotics;
the germicides;
and
the spirochaeticides.
The surprising finding of enhancement of action of the anti-infective agents referred to above is made against the background of the fact that there appears to be no earlier suggestion in the literature to the effect that either nitrous oxide or the long chain fatty acids used in the formulation referred to above, and hence also not the combination of these, has any effect whatsoever on the sensitivity of any micro-organism to any anti-infective agent.
The present invention is specifically, though not exclusively aimed at the enhancement of the action of anti-mycobacterial agents, and particularly those used in the treatment of patients infected with Mycobacterium tuberculosis (M.Tb.). This organism is one of the most significant human pathogens. It is responsible for an estimated seven million new cases of tuberculosis annually, and an estimated three million deaths worldwide. Of particular concern is the emergence of tuberculosis (TB) as an increasing cause of morbidity and mortality among persons compromised by human immune-deficiency virus (HIV) infection.
Although the prevalence of tuberculosis in developed countries declined in the first few decades of the 1900's, this trend has reversed and an increased incidence of tuberculosis has been reported in many countries. Africa alone is estimated to have approximately 170 million TB patients. In South Africa the incidence of tuberculosis is also rising and is at different levels in different population groups. The chapter on Tuberculosis in the 1999 edition of South African Health Review (available at http://www.hst.org.za/sahr/) opens with the shocking statement that: “Despite the availability of effective and affordable treatment, the number of South Africans dying from tuberculosis continues to increase”. It is echoed by the summary of the startling overview in which it is recorded that “A reported incidence of 254 cases per 100 000 for the period 1996-1998 combined with low cure rates, indicate that the epidemic is still out of control. Rising levels of HIV infection and multi-drug resistant TB (MDR TB) represent additional threats to TB control efforts.” The number of reported cases of pulmonary TB (PTB) is reported in the Review to have risen from 90628 to 110016 new reported cases per year over the period 1996 to 1998, the estimated report rate having increased over the same period from 64% to 71%. Some of the provinces in South Africa contributed significantly to the national average TB incidence figure of 254 referred to above. The quoted Review reflects the figure for Eastern Cape as 388, for the Northern Cape as 360, the Free State as 338 and the Western Cape as about 500 per 100000, almost double the national average. It has been reported elsewhere that the highest incidence of TB in the country is found in certain communities in the Western Cape where the estimated incidence is as high as 1400 per 100000.
Re-infection of patients is an ever-increasing problem and has been shown to be a function of reactivation of TB in patients not completing their therapy. It is also often associated with the appearance of drug resistant M.Tb. in the patient. The exact mechanism whereby drug resistance develops in mycobacteria is not yet fully understood, but the economic consequences thereof are a reality. The occurrence of drug resistant strains of M.Tb., generally known as multi-drug resistant Tuberculosis (“MDR TB”) is also referred to in the aforementioned South African Health Review 1999. It states: “Accurate figures for MDR TB are currently not available but surveys in three provinces (Western Cape, Mpumalanga and Gauteng) indicate a rate of 1% in new MDR TB cases, 4% in retreatment cases. This translates to at least 2 000 newly active cases of MDR TB in South Africa each year. MDR TB is extremely expensive to treat—R25 000 to R30 000 per patient for the drugs alone as opposed to less than R200 for a new patient with ordinary TB. Such patients generally also require to be hospitalised for long periods of time (usually between six and eighteen months), adding significantly to the cost of their treatment”
In an attempt to reduce discontinuance of TB-treatments which has been implicated in reinfections and the development of resistant strains, the practice of directly observed treatment or DOTS has been resorted to, with some, but based on the foregoing quotes, not complete success.
Iron, heavy metals, and excessive alcohol consumption (an inherent feature of some identified high incidence TB communities) generate harmful reactive oxygen species which have been shown to be involved in the auto-oxidation of Rifampicin, an antibiotic anti-mycobacterial agent used in the treatment of tuberculosis, thereby generating more radical species. These free radicals have been implicated in the liver toxicity experienced with use of Rifampicin.
These problems associated with TB have led to the investigations associated with the present invention.
It has also surprisingly been found that the aforesaid medium has the unexpected property that it displays a remarkable ability to enhance the action of known agents affecting the central and/or peripheral nervous system, such known agents being other than the group which consists of coal tar solution, H1 antagonist antihistamines, the analgesics, the anti-inflammatories and the antipyretics. These agents will hereinafter collectively be referred to as “CPNS agents” which term is for purposes of this specification intended to embrace those biologically active compounds which perform their action on the central or peripheral nervous system of the human or animal body, but to exclude recognised anti-inflammatory, analgesic and antipyretic substances and also excluding coal tar solution and H1 agonist antihistamines. Subject to these exclusions it therefore includes the compounds which fall within Class 1 of the Pharmacological Classification set out as part of Regulation 5(1) of the General Regulations made under The Medicines and Related Substances Control Act, Act 101 of 1965, and the active ingredients of all products falling within Class 1 of the pharmacological classification currently employed in the Monthly Index of Medical Specialities (“MIMS”) published by Times Media in South Africa, but excluding the analgesic, anti-inflammatory and anti-pyretic compounds which falls within the above two partially overlapping classes. Examples of some of the compounds falling within the group is described in greater detail below.
The exclusion of coal tar solution and H1-antagonist antihistamines, and of anti-inflammatories, analgesics and antipyretics from the ambit of the present invention is introduced without thereby conceding that the aforementioned patents and applications contain any disclosure of any CPNS agent properties of such excluded compounds, or that such properties are obvious in the light of the disclosures in such patents or applications. Such inferences are specifically denied. The exclusion is introduced simply to avoid what is anticipated to be a potential obstacle to the grant of a patent in respect of a part of the subject matter which part in itself is not considered worth contesting during examination as it might unduly delay the implementation in practice of the significant features of the present invention. It is expected that the remaining bulk of the subject matter of the present invention will greatly contribute to the accessibility of medicines for the treatment of a large range of ailments.
It has now further surprisingly been found that the aforesaid medium and other similar media have the unexpected property that it may also be used as a delivery vehicle adapted to be used for the delivery, into the nucleus of, or generally to, an animal cell, of nucleic acid compounds.
It was pointed out in WO97/17978 referred to above that nitrous oxide is a natural gas which is also produced synthetically, and also known by the trivial name “laughing gas” which has been in use for many years as an inhalation anaesthetic and analgesic, particularly in dentistry.
It was further stated that nitrous oxide has been reported to have a synergistic or potentiating effect on halothane and other gaseous anaesthetics [See Goodman & Gilman's The Pharmacological Basis of Therapeutics 8th Ed. 1990 pp. 298-300].
Since such known synergism or potentiation is based on the use of nitrous oxide administered by inhalation, and since the use of nitrous oxide on its own as an anaesthetic and analgesic has likewise been in the form of an inhalation agent, the use of nitrous oxide for all these purposes have been confined to hospitalised patients or, at best, to treatments carried out by medical practitioners in their consulting rooms, or treatments carried out by or under supervision of a nurse in charge of a home-care patient.
Nitrous oxide is known to be soluble in water and it has been reported that at 20° C. and 2 atm pressure one liter of the gas dissolves in 1.5 liters of water, see The Merck Index 10th Ed. p. 6499.
Nitrous oxide is also known for its use as a propellant gas, mainly as a substitute for propellant gases such as chlorofluorocarbons, and more particularly to produce a food product mousse such as whipped cream or chocolate mousse or quick-breaking foams for hair treatment preparations. See in this regard U.K. Patent 1033299, U.K. Patent 1105919 and European Patent Application EPA-0123827. None of these prior publications suggests that the nitrous oxide gas plays any other role than a physical one, i.e. to expand on being depressurised and thereby to create a mousse or foam. In fact it is typically regarded as an inert in these applications and useful due to the fact that it is colourless, odourless and tasteless but soluble in water and oils.
There appears to be no suggestion in the literature, other than the applicants own prior patents and patent applications referred to above, that aqueous solutions of nitrous oxide might have any effect on man or animals. As far as the present applicant knows, it has also never been suggested that nitrous oxide may be used in conjunction with any anti-infective agent to enhance the known action of such agent.
It is known in the pharmaceutical field to formulate active ingredients in so-called liposomal formulations. Unlike the present invention which is based on formulations containing long chain fatty acids and esters thereof the liposomes are based on a clearly distinguishable group of compounds namely the phospholipids, and generally also contain cholesterol as a stabilising agent and may further contain lisolecitein. These compounds or classes form no part of the present invention and, in case it is necessary to do so, are specifically excluded from the group of long chain fatty acids and derivatives thereof incorporated in the method or formulation of the invention.